1. Field of the Invention
This invention relates to methods of increasing the solubility of proteins produced recombinantly. Specifically, the invention is directed to production of recombinant proteins as fusion proteins in order to increase their solubility.
2. Related Art
1. Recombinant Protein Production
A variety of proteins of commercial value are now manufactured using recombinant DNA technology in which the DNA encoding the protein of interest is expressed in a host cell and then purified from that host cell. However, in some cases, this technology is not without problems. A number of heterologous proteins tend to aggregate in the host cell cytoplasm or periplasm when expressed recombinantly at high levels, thereby forming insoluble protein aggregate complexes commonly referred to as “inclusion bodies”. When this occurs, the inclusion bodies are first isolated from the host cell, and the protein aggregate is then solubilized.
2. Fusion Proteins
One method that has been developed to enhance the solubility of recombinantly produced proteins of interest (and, in some cases, to simplify their purification from the host cell) is to prepare the protein of interest as a “fusion protein”. To prepare a fusion protein (also known as a “chimeric protein”), the gene encoding the protein of interest can be attached to a second gene encoding a second protein, termed a “fusion partner”. In this way, a single polypeptide is produced by the host cell, and the polypeptide is comprised of the protein of interest and the fusion partner.
The fusion partner may be homologous (i.e., from the same species and/or strain as the host cell) or heterologous (i.e., from a species and/or strain other than that of the host cell) to the host cell. Examples of commonly used fusion partners include, inter alia, maltose binding protein (“MBP”), glutathione-s-transferase (“GST”), hexaHistidine (“hexaHis”) the lacZ and trpE gene products, ubiquitin, and thioredoxin. While each of these fusion partners has been demonstrated to enhance the solubility of at least one protein of interest, certain other proteins of interest do not demonstrate enhanced solubility when linked to these fusion proteins.
In certain cases, particularly where it is desirable to obtain the protein of interest in a purified form, the fusion partner and protein of interest must be separated from each other after synthesis as a single polypeptide. One means to achieve this is to provide a peptide linker between the fusion partners. This is accomplished by adding a nucleic acid molecule encoding the peptide between the gene encoding the protein of interest and the gene encoding the fusion partner. Typically, this “linker sequence” DNA encodes an oligopeptide that is a “cleavage recognition sequence” for an endopeptidase such as enterokinase, Factor Xa, or thrombin. The endopeptidase, when presented with a fusion protein containing its specific linker sequence, can thus cleave the fusion protein into its two components.
For further discussions of fusion proteins see, for example, WO 95/04076, published 9 Feb. 1995; U.S. Pat. No. 5,629,172 issued 13 May 1997; WO 94/23040, published 13 Oct. 1994; Flaschel et al., Biotech Adv., 11:31–78 (1993); European patent application 207,044, published 30 Dec. 1986; U.S. Pat. No. 5,322,930, issued 21 Jun. 1994; European Patent 293,249, published 30 Nov. 1988; U.S. Pat. No. 5,654,176, issued 5 Aug. 1997; WO 95/16044, published 15 Jun. 1995; WO 94/02502, published 3 Feb. 1994; and WO 92/13955, published 20 Aug. 1992.
3. 14-3-3 Proteins
The 14-3-3 family of proteins are acidic, highly conserved proteins with numerous isoforms. Members of this family have been found in a variety of tissues from mammals, yeast, invertebrates, and plants. The biological functions of 14-3-3 proteins are diverse, but generally appear to involve protein-protein interactions, suggesting they may generally be considered to be modulators of activity of other proteins (for reviews of this family of proteins, see Marais et al. Curr. Biol., 3:751–753 [1995]; Aiken, TIBS, 20:95–97 [1995]; Reutheret et al., Vitamins and Hormones, 52:149–175 [1996]; Wang et al., J. Mol. Evol., 43:384–398 [1996]; U.S. Pat. No. 5,597,719, issued 28 Jan. 1997).
The GF-14 proteins from Arabidopsis thaliana are members of the 14-3-3 family of proteins. Several GF-14 genes have been cloned and sequenced (Wu et al. Plant Physiol., 114:1421–1431 [1997]). One of these genes, GF-14 omega, has been shown to be expressed in E. coli as a dimer (Lu et al., The Plant Cell, 6:501–510 [1993]).
In view of the need to prepare recombinant proteins of pharmaceutical and agricultural importance in a cost-effective manner, there is a need in the art to provide novel methods of enhancing the solubility of these proteins, thereby eliminating the necessity of costly and time-consuming refolding procedures.
Accordingly, it is an object of the present invention to provide new methods of enhancing the solubility of recombinant proteins produced in bacterial host cells.
This and other such objectives will be readily apparent to the skilled artisan from this disclosure.